PROJECT SUMMARY/ABSTRACT This Small Business Innovation Research Phase II project aims to develop new targeted fluorogenic substrates capable of measuring enzyme activities in the Golgi apparatus and Endoplasmic Reticulum (ER) of living cells and tissues. The ultimate goal and the overall impact of this project is to provide an understanding of the dynamic processes that occur in intracellular enzyme and protein trafficking defects in human disease and to obtain information that will provide a pathway to more efficacious treatment options. If successful, the proposed research will provide breakthroughs needed to advance the discovery of promising new therapies and modulating drugs for neurodegenerative disorders including Gaucher disease and other lysosomal storage diseases, Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Type 2 diabetes, Lowe syndrome, Huntington's disease and allied medical conditions. In Phase I of this project, Marker Gene Technologies, Inc. established the feasibility of the technology by preparing new fluorogenic glycosidase and peptidase substrates for enzymes with localized activity in the Golgi and ER and demonstrating differential staining in living cells that are from normal or are of disease origin. In Phase II, these and additional new substrates will be evaluated for their ability to measure specific and localized inhibition or induction of enzyme activities inside individual organelles in living cells as well as to elucidate the trafficking machinery that has a functional relationship to disease progression. The new substrates and the resulting detection systems will provide innovative methods to quantitate the influence of secondary drug or protein administration on organelle-specific lysosomal, Golgi or ER enzyme activities. Furthermore, their use in screening libraries of potential drug candidates for their ability to modulate enzyme function and localization will identify new small molecule therapeutic leads based on these parameters. This makes the combined systems useful as basic research tools for a variety of significant cell biology, biochemical and medical applications. The company has engaged the collaboration of several noted academic research laboratories and research institutions as well as major pharmaceutical companies in this arena, who are eager to test the methods and systems in their existing clinical, diagnostic or drug discovery applications. The resulting assays and products will be marketed to the research, pharmaceutical, biotechnology and diagnostic industries.